Red fluorescent protein has been isolated from a Discosoma sp. and sequenced (see, e.g., Matz et al., Nature Biotech. 17:969–973 (1999), Gross et al., Proc. Nat'l Acad. Sci. USA 97:11990–11995 (2000)). A variant with humanized codons has also been engineered (Clontech, “DsRED™”). The crystal structure of red fluorescent protein has been elucidated, which demonstrated that red fluorescent protein is a tetrameric protein (Wall et al., Nat. Struc. Biol. 7:1089 (2000); Yarbrough et al., Proc. Nat'l Acad. Sci USA 16:462–467 (2000)).
Red fluorescent protein (RFP) and DsRED, as well as other fluorescent proteins such as YFP, or GFP from Aequorea victoria, Renilla reniformis, Renilla muelleri, and Ptilosarcus gurneyi, are useful are reporter molecules for a variety of bioassays, including those that use FACS as a selection mechanism (see, e.g., Tsein, Nature Biotechnology 17:956 (1999); Tsein, Ann. Rev. Biochem. 6:509–544 (1998); Heim et al., Nature 373:663–664 (1995); Heim et al., Proc. Nat'l Acad. Sci. USA 91:1250 (1994); Prasher et al., Gene 111:229 (1992); Prasher et al., Trends in Genetics 11:320 (1995); Chalfie et al., Science 263:802 (1994); and WO 95/21191). However, brighter, faster folding, and higher expressing variants would be useful.
Such variants can be made, e.g., using methods of gene shuffling and mutagenesis (see, e.g., U.S. Pat. No. 5,811,238; WO 00/73433; WO 00/22115; WO 99/41369; WO 01/04287; WO 00/46344; WO 99/45143, WO 99/41368; and Ichiro et al, Protein Science 8:731–740 (1999)). However, the use of such methods for production of variant proteins such as Discosoma red fluorescent protein variants is not always successful (see, e.g., Baird et al., Proc. Nat'l Acad. Sci. USA 97:11984–11989 (2000)). Novel methods of making such variants would therefore be useful.